Summary FDA-approved gene therapy trials have treated autosomal-recessive (i.e., loss-of-function) disorders by supplementation with the wild-type (WT) version of the mutant gene. For patients with autosomal-dominant (ad) gain-of-function disorders, the best hope for a cure is genome surgery that repairs or removes the malfunctioning genes at the root of the disease. Currently, that hope lies in CRISPR/Cas9-based gene editing {DiCarlo, Mahajan & Tsang, J Clin Invest. 2018;128:2177}. The strength of the first-generation CRISPR-based therapy (CRISPR1.0; Fig. 1)?its mutation-specificity?is also its greatest weakness. This is because the therapeutic components for each mutation (both the guide RNA (gRNA) and the repair template) need to be custom-designed, engineered, tested, and FDA-approved. This presents a considerable and costly challenge for the many ad diseases caused by a slew of different mutations. For example, the blinding Best vitelliform macular dystrophy (VMD) disorder is caused by any 1 of 250 different mutations in the rhodopsin (BEST1) gene. Treatment of all patients would, therefore, require that 250 sets of CRISPR1.0 components be engineered, validated, and FDA- approved. To overcome this major limitation, we developed CRISPR2.0 (Fig.2), a mutation nonspecific strategy. Unfortunately, CRISPR2.0 is not allele-specific and so eliminates both the mutant and WT alleles. As a result, CRISPR2.0 requires gene supplementation, which leads to variable expression of the rescued gene and sustainability concerns. We now propose to develop a third-generation CRISPR-based strategy, CRISPR3.0 (Fig. 2), that, like CRISPR2.0, is mutation nonspecific. However, CRISPR3.0 is allele-specific and therefore ablates the mutant, disease-causing cis allele while leaving the WT allele intact to support normal function. We hypothesize that CRISPR3.0 chromosome-specific genome surgery will produce a more sustained therapeutic response compared to the CRISPR2.0 supplementation strategy.